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Modulation of Cl^- signaling and ion transport by recruitment of kinases and phosphatases mediated by the regulatory protein IRBIT.

ABSTRACT: IRBIT is a multifunctional protein that controls the activity of various epithelial ion transporters including NBCe1-B. Interaction with IRBIT increases NBCe1-B activity and exposes two cryptic Cl^--sensing GXXXP sites that enable regulation of NBCe1-B by intracellular Cl^- (Cl^- _in). Here, phosphoproteomic analysis revealed that IRBIT controlled five phosphorylation sites in NBCe1-B that determined both the active conformation of the transporter and its regulation by Cl^- _in Mutational analysis suggested that the phosphorylation status of Ser²³², Ser²³³, and Ser²³⁵ was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser²³², Ser²³³, or Ser²³⁵ produced NBCe1-B in the conformations pSer²³³/pSer²³⁵, pSer²³²/pSer²³⁵, or pSer²³²/pSer²³³, respectively. The activity of the pSer²³³/pSer²³⁵ form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl^- _in The properties of the pSer²³²/pSer²³⁵ form were similar to those of wild-type NBCe1-B, whereas the pSer²³²/pSer²³³ form was partially active, further activated by IRBIT, but retained inhibition by Cl^- _in Furthermore, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control phosphorylation of Ser⁶⁵, which affected Cl^- _in sensing by the ³²GXXXP³⁶ motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser¹², which affected Cl^- _in sensing by the ¹⁹⁴GXXXP¹⁹⁸ motif. Ser²³², Ser²³³, and Ser²³⁵ are conserved in all NBCe1 variants and affect their activity. These findings reveal how multiple kinase and phosphatase pathways use phosphorylation sites to fine-tune a transporter, which have important implications for epithelial fluid and HCO₃ ^- secretion.

SUBMITTER: Vachel L

PROVIDER: S-EPMC8325875 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ameloblasts express transmembrane proteins for transport of mineral ions and regulation of pH in the enamel space. Two major transporters recently identified in ameloblasts are the Na(+)K(+)-dependent calcium transporter NCKX4 and the Na(+)-dependent HPO4 (2-) (Pi) cotransporter NaPi-2b. To regulate pH, ameloblasts express anion exchanger 2 (Ae2a,b), chloride channel Cftr, and amelogenins that can bind protons. Exposure to fluoride or null mutation of Cftr, Ae2a,b, or Amelx each results in formation of hypomineralized enamel. We hypothesized that enamel hypomineralization associated with disturbed pH regulation results from reduced ion transport by NCKX4 and NaPi-2b. This was tested by correlation analyses among the levels of Ca, Pi, Cl, Na, and K in forming enamel of mice with null mutation of Cftr, Ae2a,b, and Amelx, according to quantitative x-ray electron probe microanalysis. Immunohistochemistry, polymerase chain reaction analysis, and Western blotting confirmed the presence of apical NaPi-2b and Nckx4 in maturation-stage ameloblasts. In wild-type mice, K levels in enamel were negatively correlated with Ca and Cl but less negatively or even positively in fluorotic enamel. Na did not correlate with P or Ca in enamel of wild-type mice but showed strong positive correlation in fluorotic and nonfluorotic Ae2a,b- and Cftr-null enamel. In hypomineralizing enamel of all models tested, 1) Cl(-) was strongly reduced; 2) K(+) and Na(+) accumulated (Na(+) not in Amelx-null enamel); and 3) modulation was delayed or blocked. These results suggest that a Na(+)K(+)-dependent calcium transporter (likely NCKX4) and a Na(+)-dependent Pi transporter (potentially NaPi-2b) located in ruffle-ended ameloblasts operate in a coordinated way with the pH-regulating machinery to transport Ca(2+), Pi, and bicarbonate into maturation-stage enamel. Acidification and/or associated physicochemical/electrochemical changes in ion levels in enamel fluid near the apical ameloblast membrane may reduce the transport activity of mineral transporters, which results in hypomineralization.

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Modulation of Cl^- signaling and ion transport by recruitment of kinases and phosphatases mediated by the regulatory protein IRBIT.

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Dataset Information

Modulation of Cl- signaling and ion transport by recruitment of kinases and phosphatases mediated by the regulatory protein IRBIT.

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OmicsDI is part of the ELIXIR infrastructure

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Modulation of Cl^- signaling and ion transport by recruitment of kinases and phosphatases mediated by the regulatory protein IRBIT.